Invertebrate animal model with neurodegenerative phenotype for screening and testing substances

ABSTRACT

The present invention claims an invertebrate animal that has been modified to express a set of genes, the set comprising the gene coding for a modified version of the gamma subunit of AMP-activated protein kinase (AMPKg). According to the invention, the animal displays an identifiable phenotype related to lipid metabolism and neurodegeneration. This animal provides a model of neurodegenerative diseases, particularly Alzheimer&#39;s disease, and may be useful for screening and testing modulating agents, substances and therapeutic compounds for neurodegenerative disorders.

This is a continuation of Ser. No. 10/501,815, filed, May 11, 2005, nowabandoned which is a 371 of PCT/EP02/03992, filed Apr. 10, 2002.

The present invention relates to an invertebrate animal model ofneurodegenerative diseases with Alzheimer's disease-like pathologicalfeatures. Such animal model may be useful in screening for and testingof substances and pharmaceutical agents that modulate plaque-formationrelated to neurodegenerative diseases, particularly Alzheimer's disease.

Neurodegenerative diseases, in particular Alzheimer's disease, have aseverely debilitating impact on a patient's life. Furthermore, thesediseases constitute an enormous health, social, and economic burden.Alzheimer's disease is the most common age-related neurodegenerativecondition affecting about 10% of the population over 65 years of age andup to 45% over age 85 (for a recent review see Vickers et al., Progressin Neurobiology 2000, 60:139-165; the contents of all publications,patents and patent applications referred to and cited in the presentinvention shall be incorporated by reference in their entirety).Presently, this amounts to an estimated 12 million cases in the US,Europe, and Japan. This situation will inevitably worsen with thedemographic increase in the number of old people (“aging of the babyboomers”) in developed countries. The neuropathological hallmarks thatoccur in the brain of individuals suffering from Alzheimer's disease aresenile plaques, composed of amyloid-b protein, and profound cytoskeletalchanges coinciding with the appearance of abnormal filamentousstructures and the formation of neurofibrillary tangles. AD is aprogressive disease that is associated with early deficits in memoryformation and ultimately leads to the complete erosion of highercognitive function. Currently, there is no cure for AD, nor is there aneffective treatment to halt the progression of AD or even a method todiagnose AD ante-mortem with high probability. Several risk factors havebeen identified that predispose an individual to develop AD, among themmost prominently the epsilon4 allele of apolipoprotein E (ApoE).Although there are rare examples of early-onset AD which have beenattributed to genetic defects in the genes for APP, presenilin-1, andpresenilin-2, the prevalent form of late-onset sporadic AD is ofhitherto unknown etiologic origin. The late onset and complexpathogenesis of neurodegenerative disorders pose a formidable challengeto the development of therapeutic and diagnostic agents. Therefore, itis very important to develop suitable animal models of neurodegenerativedisease which may be useful in the development of such therapeutic anddiagnostic agents.

Although the cholesterol metabolism has long been investigated, its rolein neurodegeneration is still unclear. A connection between cholesteroland neurodegeneration has been made by the discovery that theapolipoprotein E4 (apo E4) allele is the major known risk factor forAlzheimer's disease (Saunders et al., 1993; Corder et al., 1993). ApoE4has been strongly linked to both, the sporadic as well as familiallate-onset form of Alzheimer's disease which account for approximately99% of Alzheimer's cases (Weisgraber and Mahley, 1996, Neve and Robakis,1998). ApoE is expressed in neurons and astrocytes and the firstimplication of apoE in Alzheimer's disease came from immunohistochemicalstudies, which revealed a localization of apoE in amyloid plaques andneurofibrillary tangles, the hallmarks of Alzheimer's disease (Namba etal., 1991).

ApoE is a component of several plasma and cerebrospinal fluid complexeswhich contain and transport lipids and cholesterol through theextracellular space by binding to the LDL or VLDL (low and very lowdensity lipoprotein) receptors, a transport mechanism highly conservedin vertebrates and invertebrates (Fischer et al., 1999).ApoE-cholesterol-lipoprotein complexes are assembled from freecholesterol and are internalized by neurons via the LDL receptorpathway. After uptake they are degraded and the cholesterol releasedwithin the cell were it can either be used as free cholesterol or storedin the form of cholesterol ester (Poirier, 1994; Weisgraber and Mahley,1996). The role of apoE4 in Alzheimer's disease is not yet clear but thewell established function of apoE in lipid and cholesterol transport maybe the crucial point. Studies have shown an inefficient cholesterol andphospholipid transport in Alzheimer brains resulting in an abnormalmembrane lipid composition (Koudinova et al, 1996). In addition, thepathogenic Aβ peptide, which is produced from the amyloid precursorprotein (APP) by cleavage through β-secretase, decreases cholesterolesterification and changes the distribution of free cholesterol inneurons (Koudinova et al., 1996; Liu et al., 1998). On the other side,the cleavage of APP by secretases is depending on the level ofcholesterol (DeStrooper and Annaert, 2000). The cholesterol synthesis inneurons is regulated by hydroxy-methylglutaryl-CoA reductase (HMG-CoA).An inhibition of this enzyme not only reduces the cellular cholesterollevel but also inhibits β-secretase cleavage of APP (Frears, et al,1999). HMG-CoA activity is negatively regulated via phosphorylationthrough the AMP-activated protein kinase (AMPK), a heterotrimericcomplex, consisting of the catalytic a subunit and a b and g subunit,found in all eukaryotes (Hardie et al., 1998, Kemp et al., 1999; GenBankaccession numbers of the nucleotide sequence of Drosophila AMPKg:NM080509, AF094764; GenBank accession number of the protein sequence forDrosophila AMPKg: NP536757, AAC95306). In addition AMPK inhibits theactivation of hormone-sensitive lipase, an enzyme involved in thebreakdown of triglycerides and cholesterol ester (Garton et al., 1989).

It is an object of the present invention to provide an animal modeluseful for the screening and testing of modulating agents ofneurodegenerative diseases, particularly Alzheimer's disease. Non-humanmammalian animal models, such as primates and mice, etc., are expensive,may be difficult to use, suffer from a slow reproduction time with thegeneration of only small numbers of offspring, and require a lengthyperiod of time until late onset neurodegenerative symptoms andphenotypes can be observed. Therefore, there is a need for novel animalmodels having comparatively rapid reproduction cycles with large numbersof offspring. Such an animal would offer the advantage of simple andeconomic handling and would be ideally suited for screening and testingof modulating agents of neurodegenerative diseases. Of particularinterest for this purpose are invertebrate animal models, in particularan invertebrate transgenic animal model of the fruit fly Drosophilamelanogaster (see Fortini et al., 2000). Based on the surprising findingthat the Drosophila mutant loechrig (loe), which is caused by atransposon insertion in the gene for the AMPK g subunit, ischaracterized by a low level of cholesterol ester together with strongneurodegenerative features, the present invention further providesmethods and applications useful for the identification and testing ofmodulators, compounds and therapeutic agents for neurodegenerativediseases, particularly Alzheimer's disease. Furthermore, based on thegenetic interaction of loe with beta amyloid protein precursor-like(Appl) which results in an aberrant processing of APPL, the presentinvention features a model system and methods of screening forsubstances that modulate the proteolytic processing of APP.

The singular forms “a”, “an”, and “the” as used herein and in the claimsinclude plural reference unless the context dictates otherwise. Forexample, “a cell” means as well a plurality of cells, and so forth. Theterm “and/or” as used in the present specification and in the claimsimplies that the phrases before and after this term are to be consideredeither as alternatives or in combination. For instance, the wording“determination of a level and/or an activity” means that either only alevel, or only an activity, or both a level and an activity aredetermined. The term “level” as used herein is meant to comprise a gageof, or a measure of the amount of, or a concentration of a transcriptionproduct, for instance an mRNA, or a translation product, for instance aprotein or polypeptide. The term “activity” as used herein shall beunderstood as a measure for the ability of a transcription product or atranslation product to produce a biological effect or a measure for alevel of biologically active molecules. The term “activity” also refersto enzymatic activity. The terms “level” and/or “activity” as usedherein further refer to gene expression levels or gene activity. Geneexpression can be defined as the utilization of the informationcontained in a gene by transcription and translation leading to theproduction of a gene product. A gene product comprises either RNA orprotein and is the result of expression of a gene. The amount of a geneproduct can be used to measure how active a gene is. The term “gene” asused in the present specification and in the claims comprises bothcoding regions (exons) as well as non-coding regions (e.g. non-codingregulatory elements such as promoters or enhancers, introns, leader andtrailer sequences). The term “fragment” as used herein is meant tocomprise e.g. an alternatively spliced, or truncated, or otherwisecleaved transcription product or translation product. A “modifiedversion” of a gene can be understood as a fragment of a gene, or analternative splice variant, or a gene comprising a modified nucleic acidsequence, said modified nucleic acid sequence comprising deletions,insertions, inversions, or mutations. The term “derivative” as usedherein refers to a mutant, or an RNA-edited, or a chemically modified,or otherwise altered transcription product, or to a mutant, orchemically modified, or otherwise altered translation product. Forinstance, a “derivative” may be generated by processes such as alteredphosphorylation, or glycosylation, or lipidation, or by altered signalpeptide cleavage or other types of maturation cleavage. These processesmay occur post-translationally. The term “modulator” as used in thepresent invention and in the claims refers to a molecule capable ofchanging or altering the level and/or the activity of a gene, or atranscription product of a gene, or a translation product of a gene.Preferably, a “modulator” is capable of changing or altering thebiological activity of a transcription product or a translation productof a gene. Said modulation, for instance, may be an increase or adecrease in enzyme activity, a change in binding characteristics, or anyother change or alteration in the biological, functional, orimmunological properties of said translation product of a gene. The term‘AD’ shall mean Alzheimer's disease.

Neurodegenerative diseases or disorders according to the presentinvention comprise Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, Pick's disease,fronto-temporal dementia, progressive nuclear palsy, corticobasaldegeneration, cerebro-vascular dementia, multiple system atrophy, andmild-cognitive impairment. Further conditions involvingneurodegenerative processes are, for instance, ischemic stroke,age-related macular degeneration, and narcolepsy.

In one aspect, the present invention features a non-human animal thatexpresses a modified version of the gene coding for the gamma subunit ofAMP-activated protein kinase (AMPKg). In a preferred embodiment, saidanimal is an invertebrate. Preferably, said animal is an insect, inparticular a fly. The animal is preferably obtainable by a methodselected from the group consisting of transposon insertion mutagenesisand chemical mutagenesis of the gene coding for the gamma subunit ofAMP-activated protein kinase (AMPKg). It is preferred that said modifiedversion of the gene coding for the gamma subunit of AMP-activatedprotein kinase (AMPKg) is the loechrig (loe) mutation.

In a further preferred embodiment, the expression of said gene resultsin an identifiable phenotype in said animal. The identifiable phenotypeis related to lipid metabolism and/or is a neurodegenerative phenotype.

It is another preferred embodiment that the animal according to thepresent invention expresses a gene coding for an amyloid precursorprotein, or a modified version thereof, in particular a fragment or amutant thereof. It is desirable that said modified version of the genecoding for an amyloid precursor protein is a modified version of thegene coding for beta amyloid protein precursor-like (Appl) protein. Itis further desirable that said modified version comprises a deletion, ora partial deletion, of the gene coding for beta amyloid proteinprecursor-like (Appl) protein, wherein said deletion, or partialdeletion, results in a loss-of-function of said gene. In one embodimentof the present invention, it may be desirable that said animal istransgenic for a modified version of the gene coding for the gammasubunit of AMP-activated protein kinase (AMPKg) and/or a gene coding foran amyloid precursor protein, or a modified version thereof, inparticular a fragment or a mutant thereof.

The animal, according to the present invention, is useful foridentifying a modulator which affects lipid metabolism. It is furtheruseful for identifying a modulator which affects a neurodegenerativephenotype. Another use of an animal according to the present inventionis for identifying a modulator which affects processing of an amyloidprecursor protein.

The present invention provides a method of identifying a modulator oflipid metabolism, and/or neurodegenerative phenotype, and/or amyloidprecursor protein processing, comprising administering a substance, or aplurality of substances, to said animal; and observing the effect ofsaid substance, or plurality of substances, on said animal. In apreferred embodiment, said substance, or plurality of substances, isorally administered to said animal.

In another aspect, there is provision for the use of an animal accordingto the present invention for identifying whether a gene, or a mutantthereof, is capable of modulating a phenotype related to lipidmetabolism and/or neurodegeneration, in particular processing of anamyloid precursor protein. The modulation can be a suppression or anenhancement of said phenotype and/or processing of an amyloid precursorprotein.

Other features and advantages of the invention will be apparent from thefollowing description of figures and examples.

FIG. 1. Phenotype of the loe mutant. (A-E) Horizontal plastic brainsections stained with toloudine blue. (A) Brain sections from 1.instarlarvae do not reveal any signs of degeneration. (B). In 3.instar larvavacuoles are forming in the active areas of the ventral ganglion (vg)and the central part of the brain (cb), while the newly forming opticsystem (ol) is free. (C) Sections through the brain of a stage P8 pupaappear wild type in contrast to brain sections from a pupa shortlybefore eclosion (D) where first vacuoles are visible (arrows). (E) A 10d old loe fly shows massive degeneration compared to (F) wild type. re,retina. Bar in A 15 μm, in B-D 50 μm.

FIG. 2. Accumulation of fatty acids and necrotic cell death in loe. (A)EM brain sections from 7 d old loe flies reveal fatty inclusions(arrows). (B) The fatty acids seem to originate from within the cellbecause they are surrounded by residual cell cytoplasm (arrowheads),including a mitochondrium (mt). (C) Dying neurons, in this casemonopolar cells of the optic system, in 7 d old loe flies show thecharacteristic swelling and lysis of necrotic cell death, while thenucleus (nu) stays intact. (D)Wild type monopolar cells. Bar 2 μm.

FIG. 3. Structural analysis of the loe gene. (A) Genomic region adjacentto the P-element (PlacW). The exon-intron structures of the various loetranscripts are shown underneath. Start codons are indicated by arrows.The deletion loe^(D1) is indicated by a striped bar. B=BamH1, S=SstI,X=XbaI, C=ClaI. (B) The homology to other AMPK g-subunits is restrictedto the C-terminus (only loeI shown), including thecystathionine-b-synthase domains (light gray). The identity is givenabove. The N-terminal fragment of LoeI (dark gray) shows homology to therat X11a protein.

FIG. 4. Expression of loe mRNA. (A) Some transcripts, including threealso detected with a probe derived from exon 1-3 of loeI (arrows and B)are specific or more abundant in heads compared to bodies of w¹¹¹⁸flies. (B) Analysis of these transcripts reveals a largerfusion-transcript for the strongly expressed 4.7 kb form (arrow) in theloe mutant, which is also recognized by a P-element specific probe (C)(lacZ and white are transcripts encoded by the P-element, arrowheads).(D) The expression of loeII is unaltered. w¹¹¹⁸ was used as controlbecause this line provides the same genetic background as the mutant.rp49 was used as loading control.

FIG. 5. loeI expression rescues the phenotype. Paraffin sections from 14d old flies reveal the characteristic loe phenotype in (A) a control loefly carrying only the UAS-loeI construct without the Gal4-driverconstruct. (B) Wild type. (C) Expressing loeII in neurons with elav-Gal4does not rescue the phenotype. (D) In contrast, expression of loeI inneurons completely restores the wild type phenotype in loe. (E) Aconstruct deleting the first 738aa of loeI, while leaving the conservedC-terminus intact, shows only partial rescue ability. (F) The constructwithout the X11a similar domain (deleting aa 1-319) reveals a better butstill incomplete rescue, because some vacuoles are forming (arrows). re,retina; cb, central brain; ol, optic lobes. Bar 50 μm.

FIG. 6. Genetic interaction between loe and Appl. (A) A 4 d old Appl^(d)mutant reveals no vacuolization, whereas loe at the same age displaysmany vacuoles (B). (C) In a 4 d old Appl^(d); loe double mutant thisphenotype is enhanced and more and larger vacuoles are formed. Inaddition female Appl^(d); loe flies are sterile and have small ovaries(D) compared to wild type (E). Bar in A-C 50 μm, in D-E 150 μm.

FIG. 7. Aberrant processing of APPL in loe. The APPL antiserum revealsin Western-blots, besides some unspecific bands, three specific APPLbands which are missing in the Appl mutant. Whereas the amount of theAPPL precursor protein of 145 kDa is similar in loe flies compared tothe control line w¹¹¹⁸, the amount of the secreted form of 135 kDa isdecreased (upper arrow). The small form of 61 kDa is completely missingin loe mutants. The loading control, using an anti rasGAP antibody, isshown underneath.

EXAMPLE 1

Degeneration in Loe is Restricted to Differentiated Neurons:

The line löchrig (loe) was identified and isolated from a collection ofP-element insertion lines from Deak et al. (1997). Approximately 800lines which revealed a shortened adult life span were aged and screenedhistologically for signs of neurodegeneration. Two of these lines, withinsertions at the same position, showed massive vacuolization of thecentral nervous system a few days after eclosion, which increased withaging. Vacuoles are most prominent around the central complex and in thecentral parts of the brain whereas the optic lobes are less affected(FIG. 1E). Electron microscopic analysis revealed the accumulation of afatty substance (FIG. 2A), presumably unsaturated fatty acids due to thestabilization by osmium (Ruthmann, 1966), which originates in the cellcytoplasm (FIG. 2B). To determine whether the degeneration is restrictedto the adult CNS we examined various developmental stages for signs ofdegeneration and abnormal cell death. We could not detect any vacuolesor other degenerative defects in the brains from 1. instar larvae,suggesting that the embryonic development is undisturbed (FIG. 1A). In3. instar larvae however vacuolization is clearly visible in thehemispheres as well as the ventral ganglia of the CNS. Interestingly,the degeneration is restricted to the central half of the hemispheres(FIG. 1B), an area which shows neuronal activity in larvae. In contrast,the newly developing optic system is free of vacuoles. Examinationduring pupal development revealed that pupal brains from stage P8(Bainbridge and Bownes, 1981) were free of vacuoles (FIG. 1C) whereaspupae shortly before eclosion (P15) showed first vacuoles in the centralbrain (FIG. 1D, arrows). These results indicate that the vacuolizationand degeneration in loe is confined to differentiated, probably synapticactive neurons, whereas neuroblast and developing neurons areunaffected.

To assess whether dying cells undergo apoptotic or necrotic cell deathwe performed TUNEL stainings (Gavrieli et al., 1992) and electronmicroscopic studies. The observed swelling and lysis of cell bodies,while the nucleus stays intact, are characteristic features for anecrotic cell death (FIG. 2C), which is also supported by the negativeTUNEL staining on head cryosections. In addition, the EM sectionsconfirmed that the dying cells are neurons because glial cells appearedmorphologically wild type.

Loe Encodes a Subunit of the AMP Dependent Protein Kinase Complex:

To verify that the mutation is caused by the insertion of the P-elementwe remobilized the P-element (O'Kane, 1998) to restore the wild typephenotype. From 100 established lines 95 showed a reversion of thevacuolization phenotype in paraffin head sections. 30 of these lineswere characterized in more detail, revealing a precise excision of theP-element in two of the revertant lines. These results confirm themutagenic effect of the P-element, which was consequently used toisolate neighboring genes via plasmid rescue (O'Kane, 1998).

We could isolate approximately 20 kb genomic DNA adjacent on either sideof the P-element insertion site performing plasmid rescues. Within thisregion we found homology to a first cDNA fragment from the BerkeleySequencing Project and to genomic clones from the Drosophila GenomeProject. Various other cDNAs were isolated by their homology to eitherof these clones. Their further characterization revealed that theyrepresent at least six alternatively spliced transcripts for theDrosophila gamma subunit of AMP activated protein kinase (AMPK) (FIG.3A). The different mRNAs encode at least three different proteinisoforms, all sharing the same C-terminus while varying in theirN-terminal part. The C-terminus includes the so-called CBS(cystathionine-b-synthase) domains which are highly conserved betweenthe Drosophila, yeast and mammalian proteins (FIG. 3B). Interestingly, aregion in the unique N-terminus of the LoeI isoform shows homology tothe X11a protein which can bind to the Ab peptide of APP (Borg et al.,1998); loeI and X11a are 28% identical and 41% similar over a stretch of80 amino acids (FIG. 3B). The P-element is inserted in the seventhintron of this transcript and 38 bp upstream of the transcription startsite of loeII (FIG. 3A), suggesting that one or both transcripts areaffected by the insertion (all other transcripts are localized more than10 kb downstream of the insertion site and are therefore most likely notaffected by the P-element). We created a small deletion of 1.3 kb aroundthe insertion site, removing exon 1 of the loeII transcript (FIG. 3A,loe^(D1)) and these flies do not show a degeneration phenotype. Thisindicates that loeII is not required for CNS integrity.

The Mutation is Due to an Aberrant LoeI Transcript:

Northern blot analysis of adult body and head mRNA fractions furthersupported that the mutation is due to an effect on the loeI transcript.Using a probe complementary to the conserved 3′ ends of loe revealsseveral transcripts, some of them enriched in heads compared to bodies(FIG. 4A). A probe comprising exon 1-3 from loeI detected three of thesetranscripts (FIG. 4A, arrows, 4B), with a size of 7.6 kb, 4.7 kb (thesize of the cDNA), and 0.7 kb. Comparing transcripts in head homogenatesfrom wild type and loe mutant flies revealed a change of only the 4.7 kbloeI transcript, increasing it in size to approximately 5.5 kb in themutant (FIG. 4B, arrow). The hybridization of this aberrant transcriptwith a P-element specific probe proves that it is due to splicing partsof the P-element into the loeI transcript (FIG. 4C). Other transcripts,including loeII, are not altered in mutant flies (FIG. 4D).

To finally confirm the role of loeI, we expressed the loeI and loeIIcDNA in different cell types using the UAS/Gal4 system (Brand andPerrimon, 1993). Lines carrying P-element vectors with either the loeIor loeII cDNA under the control of the Gal4 binding sequence (UAS) werecrossed with various Gal4 lines to induce expression of loe in differentcell types. A rescue of the loe phenotype could only be achieved byusing the neuron specific elav-Gal4 line (Luo et al., 1994) incombination with UAS-loeI (FIG. 5D). Expression in glia with theloco-Gal4 line (Granderath et al., 2000) did not rescue the phenotypenor did expression of loeII in neurons (FIG. 5C). This finally provesthat the mutation is caused by a disruption of only the loeI transcript.In addition, these experiments reveal a requirement for this transcriptin neurons because glial expression can not rescue the phenotype.

The Unique N-Terminus of LoeI is Required for Wild Type Function:

To further investigate the function of the transcript specificN-terminal region we created shortened loeI constructs. Expression inneurons of a construct deleting aa 1-738, leaving the conservedC-terminus intact, could only partially improve the phenotype (FIG. 5E).This confirms the importance of the unique N-terminus for the specificfunction of the LoeI protein. A deletion of the X11a similar domain andthe more N-terminal part (aa 1-319) could rescue the phenotype to a muchbetter extend, however we could still detect some vacuoles (FIG. 5F).The X11a similar domain is therefore required for wild type function butother functional important domains must reside within the N-terminus ofloeI because deleting more results in a much more incomplete rescue.

Loe is Involved in Cholesterol Homeostasis:

To assess whether the loe mutation influences the cholesterolmetabolism, a role well described for AMPK (Kemp et al., 1999), wemeasured the lipid composition of fly heads. The analysis ofphospholipids and free cholesterol did riot reveal any significantdifferences between wild type and mutant flies. The amount ofcholesterol ester, however was reduced by approximately 40% (mean valuefrom 9 measurements). Expressing loeI in neurons restored the wild typelevel of cholesterol ester. This strongly suggests a connection betweencholesterol homeostasis and neurodegeneration in the loe mutant. AMPKinhibits the activation of hormone-sensitive lipase, an enzyme involvedin the breakdown of cholesterol ester in many tissues other than thebrain (Garton et al., 1989). A cholesterol ester hydrolase is alsodescribed for the brain (Gosh and Grogan, 1990), however, so far nothingis known whether this enzyme is regulated by AMPK. If a similar pathwayexists in the brain the missing inhibition by AMPK might lead to anoveractivity of this hydrolase and therefore to the reduced level ofcholesterol ester in the loe mutant.

Loe Interacts with Amyloid Precursor Protein Like:

As mentioned above cholesterol ester has been involved in the processingof Ab from APP, and also X11a has been connected with Ab. We looked forinteractions between loe and the β-amyloid protein precursor-like (Appl)gene, the fly homolog of the human APP (Rosen et al., 1989). Appl^(d)mutants, which carry a deletion in the Appl gene, (Torroja et al.,1996)alone do not reveal any signs of neurodegeneration (FIG. 6A). However,crossing Appl^(d) with loe flies shows an enhancement of thevacuolization (FIG. 6B, C). The effect is weaker in Appl^(d)heterozygous double mutants than in Appl^(d) homozygous ones. Thisreveals for the first time an involvement of an APP null allele withneurodegeneration. In addition females homozygous for both mutations aresterile and have small ovaries with only a few ovarioles (FIG. 6D),while neither loe nor Appl^(d) are sterile or have small ovaries (datanot shown). A function in ovaries has also been suggested for the humanAPP which is highly expressed in follicle cells where it may take partin membrane turnover and trafficking (Beer et al., 1995).

To determine whether this genetic interaction might be connected to anaberrant processing of APPL in the loe background we performed Westernblot analysis of brain extracts. Using an anti-APPL polyclonal antibody(Torroja at al., 1996) we detect three bands in w¹¹¹⁸, representing thegenetic background used to induce the loe mutation.(FIG. 7). The bandscorrespond to the membrane-associated precursor of 145 kDa, the 130 kDasecreted form (Luo et al., 1990) and a small form of approximately 61kDa, which are all not detectable in Appl^(d). In the loe mutant we findsimilar amounts of APPL precursor protein. However, the level of theprocessed secreted form is reduced and the small form is missingcompletely. This confirms a role of loe in APPL processing, presumablyby the alteration in cholesterol ester and the consequent changingenvironment for the membrane bound secretases. In addition it suggests aneuroprotective function of APPL, or specifically its processed forms,which are severely decreased in loe. The complete loss of APPL in thedouble mutant further enhances the neurodegenerative phenotype.

The AMP-activated protein kinase (AMPK) is a central component of aprotein kinase cascade conserved in eukaryotes (Kemp et al., 1999;Hardie et al., 1998). This enzyme acts as a metabolic sensor to monitorthe cellular AMP and ATP levels and is activated by various stresssituations such as starvation or hypoxia. AMPK modulates many aspects ofcell metabolism (Winder and Hardie, 1999). Its major function describedso far is to activate energy providing mechanisms while inactivatingenergy consuming processes in case of ATP depletion. Although the brainhas a particularly high metabolic activity nothing was known so farabout the requirement of AMPK in this tissue.

AMPK is a heterotrimer, consisting of the catalytic a subunit and a band g subunit which are required for stabilization of the complex andkinase activity. The activity of the complex is regulated byphosphorylation through an upstream kinase and both phosphorylation aswell as dephosphorylation are sensitive to AMP (Davies et al., 1995).For all three subunits different isoforms were identified which assembleto specific AMPK complexes with distinguishable tissue distribution invertebrates (Stapleton et al., 1996; Thornton et al, 1998). Whetherthese different AMPK isoforms have distinguishable physiologicalfunctions is still unclear. In contrast to vertebrates where variousisoforms are encoded by separate genes, the Drosophila g subunits arecreated by alternative splicing because we could not identify additionalgenes in the Drosophila Genome Project. Surprisingly, the Drosophila loeproteins contain domains not described in other species so far. Thatthese domains play a pivotal role for the specific function of thedifferent splice forms is shown by the rescue experiments usingdifferent splice forms and deletion constructs which do not or onlypartially restore the wild type function. In addition, the failingrescue experiments suggest that the function of the various isoformsgoes beyond the regulation of the energy demand and very likely the sameapplies to vertebrate isoforms.

The vacuolization in loe is accompanied by the accumulation of fattyacids. One of the targets identified for AMPK is acetyl-CoA carboxylasewhich catalyzes a key step in fatty acid synthesis. It has been showthat activation of AMPK inhibits fatty acid synthesis (Hardie et al.,1998). This suggests of course an increased fatty acid synthesis whenAMPK is inactive, and the accumulation of fatty acids in loe istherefore in good agreement with the role of AMPK in fatty acidsynthesis in cell culture.

AMPK has a central role in the cholesterol metabolism by regulatingHMG-CoA reductase which is the key regulator for the biosynthesis ofcholesterol, and hormone-sensitive lipase which is involved in thebreakdown of cholesterol ester (Garton et al., 1989). Both arenegatively regulated by AMPK which can therefore inhibit the synthesisof cellular cholesterol as well as control the storage and recycling inform of cholesterol ester. The reduced level of cholesterol ester in loemutants confirms the influence of AMPK on cholesterol homeostasis shownin cell culture (Hardie et al., 1998, Kemp et al., 1999). The effect oncholesterol ester, while free cholesterol is unaffected, could be due toa specific influence of loe on the regulation of a cholesterol esterhydrolase like hormone-sensitive lipase. However, free cholesterol mightbe effected as well, but a decrease could rapidly be counterbalanced byrecycling from cholesterol ester. Synthesis, transport and recycling ofcholesterol is tightly connected, and therefore detailed studies have tobe done to determine which pathway is influenced directly by loe.

The present invention discloses that the loe mutation effects APPLprocessing, decreasing the amount of secreted APPL. It has recently beenshown that lowering the cholesterol concentration inhibits APP cleavageby b-secretase and interferes with the localization of APP in so-calledrafts (Simons, et al. 1998, Frears, et al. 1999). These are membranemicrodomains consisting of lipids, proteins and cholesterol and theircorrect composition seams to be required for APP processing (DeStrooperand Annaert, 2000, Drouet et al., 2000). We suggests a similar effect inour loe mutant in vivo, where an aberrant membrane composition, due tothe lowered level of cholesterol ester, decreases the production ofsecreted APPL. The loe mutant not only influences APPL processing butalso interacts genetically with the Appl^(d) null allele. Appl^(d) aswell as knock-outs of APP in mice only display subtle neurologicaldeficits (Luo et al., 1992, Müller et al., 1994, Zheng et al., 1995). Inthe background of the loe mutation we can describe for the first time aneurodegenerative phenotype for a null mutation in one of the members ofthe APP family. The enhancement of the vacuolization phenotype of loe byAppl^(d) reveals that wild type APPL and perhaps especially its secretedform, which is already decreased in loe, has a neuroprotective function.

Materials and Methods

Drosophila stocks: All stocks were maintained and raised under standardconditions. Canton S wild type and w¹¹¹⁸ were used as control stocks.

Tissue sections for light and electron microscopy: Larval brains andpupal and adult heads were prepared for light and electron microscopy asdescribed in Kretzschmar et al. (1997). For light microscopy, 1 μmserial sections were cut and stained with 1% toluidine blue, 1% Borax.Ultrathin Epon plastic sections were postfixed with osmium and stainedwith 2% uranyl acetate, followed by Reynolds' lead citrate (Reynolds,1963), and stabilized for transmission electron microscopy by carboncoating. Examination was done with a Zeiss EM10C/VR electron microscopeat 40-80 kV. Parrafin mass histology was performed as described by Jägerand Fischbach (Ashburner, 1989).

Cloning and sequencing: The cDNA clones for the various loe transcriptsand genomic clones were isolated from the Drosophila Genome Project(cDNAs:# SD02114, LD45665, LD28468, SD02088, GH16589, LD19285, LD41424,GH28591, LD05242, LD13337 and GH08914). The pIndy5 (kindly provided byL. Seroude) and pCaSpeR3-UAS (pUAST, Flybase) vectors were used for thepUAS-loe constructs. Sequencing was performed using the Thermo Sequenasefluorescent labeled primer cycle sequencing kit from Amersham Pharmaciaafter subcloning cDNA fragments into pBluscript KS. Reactions were doneon a Hybaid Omn-E (MWG) thermocycler according to the instruction manualfor the sequencing kit. Sequence analysis followed with the ALFexpresssequencing system (Pharmacia) using Hydrolink Long Ranger gels (FMC BioProducts).

Northern blots: Total RNA was isolated using the Trizol method describedin Goodwin et al., 1997 and poly mRNA selected with the PromegaPolyAtract system. Northern blots were performed following the protocolof Ausubel et al., 1996.

Lipid and sterol measurements: 2 mg of fly heads were homogenizedmechanically and chloroform/methanol extracted as described in Folch etal., 1957. Phospholipids were separated by two-dimensional thin-layerchromatography on Silica gel 60 plates (Merck) usingchloroform/methanol/25% NH3 (65:35:5; per vol.) andchloroform/acetone/methanol/acetic acid/water (50:20:10:10:5; per vol.)as solvents. Phospholipids were visualized on TLC plates by stainingwith iodine vapor, then scraped off and quantified (Broekhyse, 1968).For the analysis of neutral lipids, extracts were applied to Silica gel60 plates with a sample applicator (Linomat IV; CAMAG) and chromatogramsdeveloped in an ascending manner using the solvent system lightpetroleum/diethyl ether/acetic acid (70:30:2; per vol.). Quantitation ofsterol and sterol ester was carried out by densitometric scanning at 275nm with ergosterol as standard. Neutral lipids were visualized bypost-chromatographic staining using a chromatogram immersion device(CAMAG). Quantitation of sterol and sterol esters was carried out bydensitometric scanning at 275 nm with ergosterol as standard.Quantification of triacylglycerols, sterol and sterol ester was carriedout by densitometric scanning at 400 nm with triolein.

Western blot analysis: Fly heads were homogenized as described inTorroja et al., 1996 and loaded on 7.5% SDS-PAGE gels using standardmethods (Laemmli, 1970). Proteins were transferred onto nitrocellulosemembranes (Towbin et al., 1979). Immunoreactions with anti-APPL (Ab952,kindly provided by K. White), diluted 1:300 and preadsorbed over nightagainst Appl^(d) embryos, was done following the manufacturer's protocolfor ECL Western Blot Detection System (Amersham). Hybridoma supernatantwas diluted 1:3 for detecting rasGAP as the loading control.

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1. A transgenic Drosophila whose genome is homozygous for the loechrig(loe) allele and homozygous for the Drosophila β-amyloid precursor-likedeletion allele (Appl^(d)), wherein said transgenic Drosophila exhibitsenhanced vacuolization, sterility in female Drosophila,neurodegeneration, accumulation of fatty acids in the brain andreduction of cholesterol ester.